Listeria monocytogenes is a major food-borne pathogen that is the cause of significant morbidity and mortality worldwide. It is also currently in clinical trials as an anti-cancer therapeutic agent. Many of the molecular details of the pathogenesis of L. monocytogenes have been revealed, and it is one of the most comprehensive models of immunity. Nonetheless, much remains to be discovered, particularly in regard to host pathogen interaction in the live animal or human subject. We have recently identified the gallbladder as a potential reservoir of bacteria in symptomatic and asymptomatic infected mice, and have determined that its replication in this tissue is extracellular, whereas intracellular replication is the primary pattern of growth and pathogenesis of this bacterium in vivo. Replication in the gall bladder could be the source of asymptomatic carriage of this pathogen in humans and a potential reservoir leading to food contamination. Here we seek to further characterize the genetics and unique physiology of Listeria in the gallbladder as a means to preventing food contamination and improving antimicrobial therapies. We propose three specific aims. First, we will perform a quantitative image-guided analysis of intracellular replication of L. monocytogenes in the murine gallbladder. Our previous results demonstrate that replication in the gall bladder does not require prototypical virulence factors, suggesting that virulence mediated by intracellular replication is distinct from growth in the gall bladder. Mutations of the prfA gene, the central control element of listerial virulence, do not affect the ability to grow in the gall bladder. The major bile resistance locus bilE is dispensable for replication in the gallbladder. These results suggest a unique set of genes for infection of this site, thus we propose a comprehensive and quantitative functional genomics analysis. Second, we will determine the gene expression patterns of L. monocytogenes in the gallbladder lumen and in pure bile culture in comparison to the pattern observed in ordinary culture. We hypothesize that patterns of gene expression will closely resemble, but be distinct from, those induced in pure bile culture. Mutants of highly induced gene will then be constructed. Lastly, we will measure the composition of gallbladder bile removed from infected and uninfected animals and from pure bile cultures using mass spectrometry. We will use mass spectrometry of luminal contents to assess the status of bile components in the infected gallbladder, and determine the effects of bacterial growth on bile composition in pure bile culture. We hypothesize that the bacterium will alter the bile components of the lumen and in pure bile culture, to avoid bile toxicity and obtain nutrients. The mutants constructed in the Second Aim, will then be employed in the assays of the First and Third Aims, to characterize the genetics and bacterial physiology of replication in the gallbladder. Public Health Relevance: The bacterium Listeria monocytogenes is one of the best understood infectious agents and has provided critical information about how bacteria infect people. We have discovered that this bacterium grows inside the gallbladder, which is highly unusual and unique in any animal model, and resembles human typhoid fever. We propose to use molecular imaging techniques to learn how this bacterium colonizes the gallbladder, constituting the only current animal model of the human carrier state of typhoid fever.